The early lesions of atherosclerosis, in humans subjects and experimental animals, consistently develop in a non-random pattern in the arterial vasculature, and the geometry of these "lesion-prone areas" correlates with branch points, curvatures and other regions of altered blood flow. Previous studies in our laboratory have demonstrated direct effects of biomechanical forces on the expression of pathophysiologically relevant genes by the endothelial cell (EC). This has led us to hypothesize that hemodynamic forces, in particular wall shear stresses generated by steady laminar and disturbed laminar flow, can function as both positive and negative stimuli in atherogenesis, via effects on EC gene expression. To test this hypothesis, we propose a series of interrelated in vitro and in vivo experiments, utilizing a combination of cell biological, molecular biological and experimental pathological techniques under three Specific Aims. In the First Specific Aim, we will extend and critically test this hypothesis (established in principle during the previous project period) by utilizing a newly developed "arterial waveform" flow device to simulate the fluid mechanics of pulsatile human blood flow on cultured human endothelial cells in vitro, and define patterns of gene expression via high-throughput transcriptional profiling techniques and analytical methods previously developed in this project. In the Second Specific Aim, we will validate in vivo, patterns of expression of putative pathophysiologically relevant endothelial genes, identified via sentinel cluster analysis of in vitro transcriptional profiling data in Specific Aim 1, directly in "high-probability" and "low-probability" regions of the mouse aorta, in atherosclerosis-prone strains, and in appropriate normal and diseased human arterial tissue samples. In the Third Specific Aim, we will critically test the hypothesis that shear-regulated endothelial gene expression contributes to athero-susceptibility, using various transgenic strategies to modulate expression of candidate pathogenic and/or protective shear-regulated genes in murine models of atherosclerosis, and observe the effects on lesion localization and progression/regression. The proposed studies thus focus on a fundamental aspect of the pathobiology of atherosclerosis - the role of biomechanical forces as a determinant of the geometric pattern of lesion formation. The combination of high-throughput genomics with sentinel-cluster analysis, and in vitro modeling with in vivo validation, promises to provide new insights into disease mechanisms.